The peroxisome, an essential subcellular compartment in all eukaryotic cells, is infimately involved in lipid metabolism. Work in multiple model systems has uncovered at least 32 PEX genes encoding peroxins, that play roles In peroxisome biogenesis, morphogenesis (size, volume and number), and inheritance. However, despite this wealth of knowledge regarding the proteins involved, the biochemical functions and mechanisms of acfion of most peroxins are pooriy understood. Peroxisomal proteins are targeted to the organelle matrix by two peroxisomal targefing signals, PTS1 and PTS2. PTS receptors bound to their cargo interact with the importomer, a complex of peroxisome-membrane-assoclated Pex proteins. The importomer, responsible for peroxisomal matrix protein translocation is comprised ofthe docking and RING subcomplexes. During the matrix protein import cycle, the receptor-cargo complexes shuttle from the cytosol, interact with the importomer, release cargo in the peroxisome matrix and the receptors then shutfie back to the cytosol, aided by a receptor-recycling subcomplex. Major unanswered questions in the peroxisome biogenesis field relate to the exact nature, function and structure ofthe unusual peroxisomal translocon which, unlike translocons associated with other subcellular compartments, transports folded and oligomeric proteins across (Aimi). The evolution of dual matrix protein import pathways and the steps in cargo release and receptor recycling from peroxisomes. Involving the function of Pex8, are critical for an understanding of the import cycle (Aim 2). The recent emergence of a clear involvement of the endoplasmic reficulum (ER) in peroxisome morphogenesis, and even biogenesis, raises important quesfions about the machinery that sorts peroxisomal membrane proteins via the ER and the funcfional role of this pathway in peroxisome growth and division (Aim 3). Finally, we wish to further explore fascinating emerging clues regarding a crosstalk between peroxisome biogenesis, inheritance and turnover (Aim 4). We believe we are uniquely positioned to answer these fundamentally important cell biological quesfions that seriously impact human health and disease. RELEVANCE (See instructions): Impairment of peroxisomal metabolic or biogenesis pathways causes 17, often fatal, human disorders. This proposal will address basic mechanisms involved in peroxisomal matrix and membrane protein assembly, growth and division. By doing so we hope to understand how organelle homeostasis is maintained and how the impairment of these processes can affect human health and disease.